User:Paul Rothenberg/Notebook/CHEM 571 Fall 2014/2015/02/12

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Kinetics of 1 nM Proteinase K Degradation of AuNP Fibers

Objectives

To continue gathering kinetics data for the protease reaction.

Protocol

1 nM Proteinase K was made by taking 10 µL of the stock, and diluting up to 1 mL with buffer. 6.9 µL of that was diluted up to 3 mL with buffer for a final reactant volume concentration of 1 nM protease. The fibers were spun at 300 RPM for 10 minutes, and the liquid extracted. The solid fibers were added to the cuvette with minimal liquid. The protease was added on top.

The OceanOptics was set at 37°C, scan every 5 minutes for 6 hours.

Results

Based on visual analysis after the 6 hour period, the fibers were still present in solution, and there was not much of a discernible colorimetric shift. There was also particulate matter on top of the cuvette when the reaction finished.

1nM ProtK Kinetics AbsvsTime Chart.png

1nM ProtK Kinetics Chart.png

February 18th, 2015

Trypsin Reaction Preparation

0.00077 g of Trypsin was dissolved in 1 mL of Tris/NaCl buffer ---> stock [trypsin] = 33.05 μM


Bradford/Gel Analysis samples to be run for three/four hours; extracting 500 μL samples every 30 minutes:

1. 0.151 mL of stock solution in 4.849 mL of Tris/NaCl buffer

  • [trypsin]final = 1 μM

2. 0.015(1) mL of stock solution in 4.985 mL of Tris/NaCl buffer

  • [trypsin]final = 100 nM

3. 0.001(5) mL of stock solution in 4.998 mL of Tris/NaCl buffer

  • [trypsin]final = 10 nM

4. 10 μL of stock solution in 990 μL of Tris/NaCl buffer --> [trypsin] = 0.3305 μM

    • Take 0.015(1) mL of dilution in 4.985 mL of Tris/NaCl buffer
  • [trypsin]final = 1 nM

Fibers were spun down at 300 RPM for 5 minutes, and liquid extracted. 5 mL of protease solution was added to each sample tube and a 500 μL aliquot from each concentration was obtained every 30 minutes beginning with time point 0. The samples were left in the incubator shaker (37C) for 3.0 hours.

  • Time points collected: 0, 30 min, 60 min, 90 min, 120 min, 150 min, 180 min


In-situ UV-Vis samples:

Today's work:

69 μL of the 0.4325 μM Proteinase K stock was diluted with 2.931 mL of Tris/NaCl buffer for a final [Proteinase K] of 10 nM. This was added onto the fibers in the cuvette, which were spun down at 300 RPM for five minutes, liquid extracted, and resuspended. The reaction was run at 37°C for 6 hours, scanning every 5 minutes.

Results

The reaction did have a discernible colorimetric change. There were still fibers present in solution after 6 hours. A longer reaction may see this go to completion.

10nM ProtK Kinetics AbsvsTime Chart.png

10nM ProtK Kinetics Chart.png


ALL FROM 1:10 DILUTION - See sample 4 above!

  • The following are calculations for the future; these experiments have not been executed!

1. 0.0907 mL of diluted stock in 2.909 mL of Tris/NaCl buffer

  • [trypsin]final = 10 nM

2. 0.00907 mL of diluted stock in 2.991 mL of Tris/NaCl buffer

  • [trypsin]final = 1 nM